KR100957555B1 - Ras Fusion Protein - Google Patents

Abstract

The present invention relates to a Ras fusion protein, and more particularly, a region of N-Ras protein and a region of H-Ras protein are combined to confirm the relationship between various types of Ras protein signaling pathways and cancer development. The present invention relates to a Ras fusion protein, a gene encoding the same, and a transformant expressing the Ras fusion protein. The Ras fusion protein of the present invention can be usefully used for the development of molecular target cancer therapies that can suppress the occurrence and / or metastasis of cancer and the discovery of bio-new drugs.

Ras, fusion protein, cancer metastasis

Description

RAS Fusion Protein

The present invention relates to a Ras fusion protein, and more particularly, to a Ras fusion protein in which some regions of the N-Ras protein and some regions of the H-Ras protein are bound.

Ras protein is a 21 kDa protein that plays an important role in cell growth and differentiation. It binds to guanine nucleotides to hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). As such, Ras protein plays a very important role in cellular signaling because it acts as a molecular switch that regulates specific GTPase circuits in cells (Bourne, HR, Sanders, DA, McCormick, F., Nature, 1991, 349, 117). .

Cancer metabolic pathways vary, one of which is caused by the activation of abnormally many protein products of the 'ras' gene. It is estimated that about 30% of cancer metastases are caused by the transformation of cells into malignant phenotypes by signaling pathways of abnormally activated Ras proteins. Some cancer cells in the human body have been observed to be mutated at the 12th amino acid position of the Ras protein. When this mutation inhibits Ras protein's own GTPase enzyme activity, GTP is continuously bound and abnormal growth signals are observed. Will be delivered continuously. Carcinogenicity has been reported to be caused by abnormalities in these signaling systems. In fact, these carcinogenic ras genes are known to be closely related to pancreatic cancer, bladder cancer, lung cancer and skin cancer (Bos, J. L., Cancer Res., 49, 4682, 1989).

There are three groups of Ras genes: N-Ras (Neuroblastoma-Ras, SEQ ID NO: 1), H-Ras (Harvey-Ras, SEQ ID NO: 2), and K-Ras (Kirsten-Ras), all of which are 189 arnoacids. Encode a protein of 21 kDa molecular weight (Barbacid, 1987). The three types of Ras protein have the same amino acid sequence of 85 amino acids and the middle 80 amino acid sequence shows 85% homology, but the carboxy-terminal sequence has very low homology. Ras proteins are expressed differently in tissue-specific developmental processes, and they attach to cell membranes by binding fat to the carboxy-terminal site. It is known that palmitoylation occurs in Cys ¹⁸¹ and Cys ¹⁸⁴ after farnesylation of Cys ¹⁸⁶ in H-Ras protein, and N-Ras protein is known to have palmitoylation site only in Cys ¹⁸¹ (Fig. 1 and FIG. 2). The difference in cell membrane adhesion due to various carboxy-terminal sequences is thought to be an important factor in the cause of the differential cellular activity by the H-Ras, N-Ras, K-Ras proteins.

In a previous study, we investigated the molecular mechanisms of H-Ras selective and N-Ras selective signaling pathways on invasiveness and mobility of MCF10A cells to elucidate the role of Ras signaling pathways in cancer metastasis activity. Studies have shown that H-Ras induces invasiveness and mobility while N-Ras does not induce at all (Int. J. Cancer, 85: 176-181, 2000; Cancer Res., 63: 5454 -5461, 2003). These results indicate that H-Ras selective signaling pathway and N-Ras selective signaling pathway exhibit different biological transfer activity in MCF10A cells. In addition, previous studies on the lower signaling molecules of the Ras signaling pathway have reported that p38 MAPK is selectively activated by H-Ras, which affects the expression of MMP-2 (Cancer Res. 63: 5454-5461, 2003).

The results indicate that sites other than amino acid sequences 32-40, which are known effector binding sites in the Ras signaling pathway, are likely to affect the downstream signaling network. However, to date, no comparative analysis of the domain-selective cancer metastasis between H-Ras and N-Ras has been reported.

The present invention prepares a fusion protein in which a portion of the H-Ras protein and a portion of the N-Ras protein are combined, and a gene encoding the same, and identifies a site that affects cancer metastasis in a signaling pathway of the Ras protein. It aims to do it.

In order to achieve the above object, the present invention provides a fusion protein in which some regions of the N-Ras protein described in SEQ ID NO: 1 and some regions of the H-Ras protein described in SEQ ID NO: 2 are combined.

In the present specification, "N" refers to a mutant protein having the amino acid sequence set forth in SEQ ID NO: 3 wherein Gly, the 12th amino acid of the N-Ras protein having the nucleotide sequence set forth in SEQ ID NO: 1 is substituted with Asp.

"H" also represents a mutant protein having the amino acid sequence set forth in SEQ ID NO: 4 in which Gly, the 12th amino acid of the H-Ras protein having the amino acid sequence set forth in SEQ ID NO: 2, is substituted with Asp.

"NH" is also a fusion having an amino acid sequence as set forth in SEQ ID NO: 5, wherein the amino terminal 1-165 amino acid sequence of the protein set forth in SEQ ID NO: 3 and the carboxy terminal 166-189 amino acid sequence of the protein set forth in SEQ ID NO: 4 bind; Represents a protein.

"HN" is also a fusion having an amino acid sequence as set forth in SEQ ID NO: 6 to which the amino terminal 1-165 amino acid sequence of the protein set forth in SEQ ID NO: 4 and the carboxy terminal 166-189 amino acid sequence of the protein set forth in SEQ ID NO: 3 bind; Represents a protein.

In addition, "NCH" is the carboxy terminal 186-189 amino acid sequence (CAAX) of the mutant protein having the amino acid sequence set forth in SEQ ID NO: 3 replacing Gly, the 12th amino acid of N-Ras, with Asp, and the CAAX site of N-Ras. (CVVM) shows a fusion protein substituted with the CAAX site (CVLS) of H-Ras. In this case, "C" of CAAX represents cysteine, "A" represents aliphatic amino acid, "X" represents serine or methionine, "C" of CVVM represents cysteine, "V" represents valine, and "M" represents methionine, CVLS "C" represents cysteine, "V" valine, "L" leucine, and "S" represents cysteine. The carboxy terminal 186-189 amino acids of Ras proteins are commonly referred to as CAAX motifs.

In addition, the carboxy terminal 186-189 amino acid sequence (CAAX) of the mutant protein having the amino acid sequence set forth in SEQ ID NO: 4 in which Gly, the twelfth amino acid of "HCN" H-Ras, was substituted with Asp was replaced with the CAAX site of H-Ras ( CVLS) shows the fusion protein substituted with the CAAX site (CVVM) of N-Ras. In this case, "C" of CAAX represents cysteine, "A" represents aliphatic amino acid, "X" represents serine or methionine, "C" of CVVM represents cysteine, "V" represents valine, and "M" represents methionine, CVLS "C" represents cysteine, "V" valine, "L" leucine, and "S" represents cysteine.

In addition, "N184H" represents the mutant protein which substituted 184th amino acid of the protein shown by SEQ ID NO: 3 with leucine and cysteine.

In addition, "H184N" represents the mutant protein which substituted the 184th amino acid of the protein shown by SEQ ID NO: 4 with leucine in cysteine.

The fusion protein of the present invention is preferably composed of the same 189 amino acids as the parent protein N-Ras protein and H-Ras protein. For example, if the 1 to nth amino acid of the N-Ras protein described in SEQ ID NO: 1 is used as the amino terminus region of the fusion protein, the carboxy end region is the n + 1 to 189th of the H-Ras protein described in SEQ ID NO: 2 Amino acids are used (wherein n is a natural number of 165 or less). The fusion protein of the present invention is preferably in the form in which the amino terminal portion of the N-Ras protein and the carboxy terminal portion of the H-Ras protein are combined.

For example, the fusion protein of the present invention is preferably a form in which the amino terminal 1-120 amino acid of the N-Ras protein and the carboxy terminal 121- 189 amino acid of the H-Ras protein are combined, and the amino terminal 1 of the N-Ras protein. It is more preferred that the amino acid of the amino acid terminal 1 to 165 of the N-Ras protein and the carboxy terminal 166 to 189 of the H-Ras protein are more preferably combined with the amino acids 142 to 189 of the H-Ras protein. Most preferably, the first amino acid is in combined form. However, it will be apparent to those skilled in the art that the combination of the amino terminal N-Ras protein and the carboxy terminal H-Ras protein used in the formation of the fusion protein is not limited thereto. In addition, for the purpose of confirming the association between Ras protein expression and cancer metastasis, the fusion protein of the present invention is preferably in the form where the 12th amino acid is substituted with Asp in Gly.

In a preferred embodiment of the invention the mutant H described in SEQ ID NO: 4 to amino acids 1 to 165 of the mutant N-Ras protein having the amino acid sequence set forth in SEQ ID NO: 3 in which the 12th amino acid of the N-Ras protein is substituted with Asp. Eight different forms of fusion proteins and mutant proteins were prepared, including the NH fusion protein set forth in SEQ ID NO: 5 to which amino acids 166-189 of the -Ras protein were bound (see Figure 3).

The present invention also provides a gene encoding the fusion protein.

The gene of the present invention may vary in sequence depending on the specific configuration of the amino-terminal N-Ras protein and the carboxy-terminal C-Ras protein used in the preparation of the fusion protein, which is apparent to those skilled in the art. In addition, when there are a plurality of codons of a base capable of encoding a specific amino acid, the base sequence encoding the same amino acid sequence may vary accordingly, which is obvious to those skilled in the art. In a preferred embodiment of the present invention provides a gene having a nucleotide sequence set forth in SEQ ID NO: 7 encoding a fusion protein set forth in SEQ ID NO: 5.

The present invention also provides a recombinant expression vector comprising a gene encoding the fusion protein.

There are no particular limitations on the parent vector that can be used in the preparation of the recombinant expression vector of the present invention, and a conventional prokaryotic or eukaryotic transformation vector can be used without limitation. In a preferred embodiment of the present invention, a recombinant expression vector was prepared by inserting the gene described in SEQ ID NO: 7 into a pcDNA 3.1+ vector (see FIG. 4).

The present invention also provides a transformant transformed with the recombinant expression vector.

The transformant of the present invention can be easily prepared by introducing the recombinant expression vector into any prokaryotic or eukaryotic cell, and methods for introducing a particular vector into cells are well known to those skilled in the art. An example of such a method is a lipofectamine method. In a preferred embodiment of the present invention, a transformant in which the recombinant expression vector was introduced into MCF10A cell line, which is a breast epithelial cell, was prepared using the lipofectamine method (see FIGS. 5 and 6).

To investigate the role of Ras protein signaling pathways in cancer metastasis activity, we investigated the molecular mechanisms of H-ras selective and N-ras selective signaling on the invasiveness and mobility of MCF10A cells. Proteins induce invasiveness and mobility whereas N-Ras proteins have been reported to induce no invasiveness and mobility at all (Int. J. Cancer, 85: 176-181, 2000; Cancer Res., 63: 5454-). 5461, 2003). Based on the results of these previous studies, the inventors have determined that the amino-terminal 1-165 amino acid sequences and carboxy-terminal 166-189 amino acid sequences of N-Ras and H-Ras are in N-Ras / H-Ras form or H-Ras / N-. A fusion protein linked in the form of Ras was prepared, and a transformed cell line in which the fusion protein was introduced into human breast epithelial cells was constructed to identify which site of Ras protein is involved in metastasis of cancer.

As a result of measuring the mobility and invasiveness of each transgenic cell line, the invasion and mobility of the cells into which the fusion protein of HN and HCN were introduced decreased, whereas the invasion and mobility of the cells into which the fusion protein of NH was introduced increased (FIG. 7). And FIG. 8). This means that the CAAX domain is partly involved in the metastatic ability of breast epithelial cells induced by H-Ras protein, but the 166-189 amino acids of H-Ras protein are essential for metastatic capacity.

The produced H-Ras / N-Ras fusion protein and transformant expressing the same can be used for the development of molecular target cancer therapy for inhibiting H-Ras specific cancer metastasis and to identify the association between signal molecules related to cancer metastasis. And by discovering the action point, it can be usefully used in the treatment of cancer.

The Ras fusion protein of the present invention is useful for identifying the relationship between the signaling pathways of various types of Ras proteins and cancer development, and the development of molecular target cancer therapies that can suppress the development and / or metastasis of cancer using the same. And in the discovery of bionew drugs.

Hereinafter, the present invention will be described in more detail by way of examples.

However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.

Example  1: H- Ras / N- Ras  Preparation of fusion proteins, genes encoding them and expression vectors

<1-1> Fusion Proteins and Preparation of Genes Encoding the Same

In order to analyze the microstructure involved in H-Ras specific metastatic capacity, the inventors have found that the activated N-Ras described in SEQ ID NO: 3, wherein the 12th amino acid of N-Ras described in SEQ ID NO: 1 is changed from Gly to Asp. , Activated H-Ras described in SEQ ID NO: 4, wherein the 12th amino acid of H-Ras described in SEQ ID NO: 2 was changed from Gly to Asp, and the hypervariable carboxy terminal region of H-Ras and N-Ras (166) -189) was prepared to replace the H-Ras / N-Ras fusion protein chimera and the amino acid of the palmitoylation site or the CAAX motif interchanged with each other (see Figure 3). To this end, H-ras and N-ras genes were first isolated by PCR using pcDNA 3.1+ vectors (Guthrie cDNA Resource Center, USA) into which H-ras or N-ras genes were inserted (approximately 570 bp). snippet).

<1-1-1> H, HCN  And H184N Manufacture

H, HCN and H184N were prepared using the H-ras gene as a template. Amplification reaction for PCR is 2 μl of 10 × concentrated reaction solution, 1.5 μl of 10 mM dNTP, 1.5 μl of 25 mM MgCl _{2 ,} 1 μl of H-ras gene, 1 μl of Taq polymerase, shown in Table 1 1 μl of each forward primer, 1.5 μl of reverse primer, and 10.5 μl of distilled water were added thereto. After maintaining the reaction solution at 94 ° C. for 5 minutes, 94 ° C. 45 seconds, 55 ° C. 45 seconds, and 72 ° C. 45 seconds were repeatedly performed 30 times. Finally, the reaction solution was maintained at 72 ° C. for 7 minutes to perform polymerase chain reaction. . As a result, a gene of about 570 bp was amplified.

<1-1-2> N, NCH  And N184H Manufacture

N, NCH and N184H were prepared using the N-ras gene as a template. Amplification reaction for PCR is 2 μl of 10 × concentrated reaction solution, 1.5 μl of 10 mM dNTP, 1.5 μl of 25 mM MgCl _{2 ,} 1 μl of N-ras gene, 1 μl of Taq polymerase, shown in Table 1 1 μl of each front primer, 1.5 μl of rear primer, and 10.5 μl of distilled water were added thereto. After maintaining the reaction solution at 94 ° C. for 5 minutes, 94 ° C. 45 seconds, 55 ° C. 45 seconds, and 72 ° C. 45 seconds were repeatedly performed 30 times. Finally, the polymerase chain reaction was performed by maintaining the reaction solution at 72 ° C. for 7 minutes. . As a result, a gene of about 570 bp was amplified.

<1-1-3> NH Manufacture

The gene encoding the amino terminal 1-165 amino acid portion (about 462 bp) was amplified using the N-Ras gene as a template. Amplification reaction for PCR is 2 μl of 10 × concentrated reaction solution, 1.5 μl of 10 mM dNTP, 1.5 μl of 25 mM MgCl _{2 ,} 1 μl of N-ras gene, 1 μl of Taq polymerase, shown in Table 1 1 μl of one NH-1 front primer and 1.5 μl of rear primer and 10.5 μl of distilled water were added thereto. After maintaining the reaction solution at 94 ° C. for 5 minutes, 94 ° C. 45 seconds, 55 ° C. 45 seconds, and 72 ° C. 45 seconds were repeatedly performed 30 times. Finally, the polymerase chain reaction was performed by maintaining the reaction solution at 72 ° C. for 7 minutes. .

Next, the gene encoding the carboxy terminal 166-189 amino acid portion (about 108 bp) was amplified using H-Ras as a template. Amplification reaction for PCR is 2 μl of 10 × concentrated reaction solution, 1.5 μl of 10 mM dNTP, 1.5 μl of 25 mM MgCl _{2 ,} 1 μl of H-ras gene, 1 μl of Taq polymerase, shown in Table 1 1 μl of one NH-2 front primer, 1.5 μl of rear primer and 10.5 μl of distilled water were added thereto. After maintaining the reaction solution at 94 ° C. for 5 minutes, 94 ° C. 45 seconds, 55 ° C. 45 seconds, and 72 ° C. 45 seconds were repeatedly performed 30 times. Finally, the polymerase chain reaction was performed by maintaining the reaction solution at 72 ° C. for 7 minutes. It was.

<1-1-4> HN Manufacture

The gene encoding the amino terminal 1-165 amino acid moiety (about 462 bp) was amplified using the H-Ras gene as a template, and the gene encoding the carboxy terminal 166-189 amino acid moiety (about 108 bp) was used as the N-Ras gene. The HN gene was amplified by the same method as the NH except that it was used as a template.

Genes of each of the chimeric and mutated proteins amplified above were purified using a PCR purification kit (Promega, USA), and then dissolved in 20 μl of TE buffer and used in the next step.

<1-2> Preparation of Expression Vector

An expression vector was prepared by inserting the amplified gene into the vector (pcDNA 3.1+). The amplified gene and vector (pcDNA 3.1+) were digested by digestion with restriction enzymes BamHI and XhoI for 3 hours at 37 ° C., and then separated in 1% agarose gel. 0.1 μg of the cleaved gene and 0.1 μg vector were added to 1 μl of 10 × ligation solution and 0.5 μl ligase (Promega, USA). Distilled water was added to make the total volume 10 μl and reacted at 4 ° C. for 18 hours. An expression vector comprising the fusion protein was prepared (see FIG. 4). At this time, in the case of the H-Ras / N-Ras chimera in which the highly variable regions 166-189 of H-Ras and N-Ras are interchanged with each other, the amino terminal 1-165 amino acid portion of the H-Ras or N-Ras protein ( About 462 bp) and the carboxy terminal 166-189 amino acid portion (about 108 bp), respectively, after which the amino terminal 1-165 amino acid portion (about 462 bp) is cleaved with BamHI and EcoRV, and the carboxy terminal 166-189 amino acid portion (About 108 bp) was digested with EcoRV and XhoI and isolated in agarose gel. First, the vector was cleaved with BamHI and EcoRV to insert an amino terminal 1-165 amino acid moiety (about 462 bp), and the vector was confirmed to be inserted once again with EcoRV and XhoI, followed by a carboxy terminal 166-189 amino acid moiety (about 108 bp) was inserted to link gene fragments in H-ras / N-ras form or N-ras / H-ras form. The reaction solution was added to E. coli DH5α competent cells to transform E. coli, and then cultured in LB plate medium containing empicillin (50 µg / ml) to select E. coli transformants. Finally, the plasmid DNA of the E. coli transformant was isolated and DNA sequences were analyzed to confirm that all of the eight expression vectors prepared were modified as desired.

Example  2: Construction of Transgenic Cell Line Expressing Fusion Protein

An expression vector containing the gene encoding the H-Ras / N-Ras fusion protein prepared in Example <1-2> was introduced into MCF10A cells (Dr. D. Lowy, NCI, Bethesda, MD), which are breast epithelial cells. It was. MCF10A cells were plated at 3.5 × 10 ⁵ cells / well in 6-well plates and cultured in 80-90% confluent. The cultured cells were washed twice with PBS (phosphate-buffered saline) to remove serum, and then the lipofectamine 2000 mixed solution containing an expression vector containing the H-Ras / N-Ras chimera and mutant proteins (Invitogen, Carlsbad, Incubate for 6 hours, complete media (5% horse serum, 0.5 μg / ml hydrocortisone, 10 μg / ml insulin, 20 ng / ml EGF, 0.1 μg / ml cholera enterotoxin, 100 units /) Mld penicillin-streptomycin, DMEM / F12 medium with 2 mM L-glutamine and 0.5 μg / ml amphotericin B). After culturing for 18 hours or more, the cells were stabilized, and the cells were removed and transferred to a 100 mm culture dish, and 400 ㎍ / ml G418 medium (400 ㎍ / ml G418, 5% horse serum, 0.5 ㎍ / ml once every 2-3 days). hydrocortisone, 10 μg / ml insulin, 20 ng / ml EGF, 0.1 μg / ml cholera enterotoxin, 100 units / ml penicillin-streptomycin, DMEM / F12 medium with 2 mM L-glutamine and 0.5 μg / ml amphotericin B). I exchanged it. After culturing the cells for about a month and clones were formed, the monoclones were selected using a cloning ring. Specifically, when a clone with resistance to G418 is formed by incubating for about a month, a cloning ring is placed on each clone, and the T-xin-EDTA is added to the inside of the ring and placed in a 37 ° C. incubator until the cells fall out of the culture dish. Waited. When clones fell from the culture dish, each clone formed was transferred to a different culture dish and cultured and stored (Moon et al., Int. J. Cancer, 85, 176-181, 2000).

Then, whether the selected transformed cell lines correctly express the fusion protein and the mutant protein was confirmed by immunoblot analysis (immunoblot analysis) using an antibody that specifically binds to H-Ras or N-Ras protein. 6).

Example  3: H- Ras / N- Ras  Differential Invasiveness and Mobility of Transgenic Cells Producing Fusion Proteins

<3-1> Invasiveness study ( in in vitro invasion assay )

Invasiveness studies were conducted using a 24-well transwell unit with a diameter of 6.5 mm and a pore size of 8.0 μm (Albini et al., Cancer Res., 47, 3239- 3245, 1987). First, 0.5 mg / ml of type I collagen was coated on the outer bottom of the filter and dried for 1 hour in air, and then dried on the inner part of 0.5 mg / ml of reconstituted basement membrane material (Matrigel). At the bottom of the 24-well, 600 ml of medium containing 0.1% BSA (Sigma) was added.Inside the transwell, cells (5 × 10 ⁴ ) were placed in serum-free medium (DMEM / F12, 100 units / ml penicillin). -Streptomycin) was mixed well in 100 μl. Incubate in 37 ° C, 5% CO ₂ incubator for 17 hours, soak in methanol for 1 minute, wash, stain cell membrane with hematoxylin for 10 minutes, and then nucleate for 4 minutes with eosin. Staining was done. The inner part of the filter was carefully removed using a cotton swab to remove all remaining cells and dried at room temperature for about an hour, and then the filter was cut out and placed in xylene to remove impurities. This was placed on a slide glass, and a drop of Canada balsam was dropped on the slide glass, and the cover glass was covered and observed under a microscope. The number of cells that passed through counted at any of 13 sites under a 400-fold microscope was counted and averaged.

<3-2> Mobility Study in in vitro migration assay )

Mobility studies were conducted using a 24-well transwell unit with a diameter of 6.5 mm and a pore size of 8.0 μm. The outer part of the filter was coated with 0.5 mg / ml Type I colarps and dried in air for 1 hour. 600 ml of medium containing 0.1% BSA is placed at the bottom of the 24-well, and cells (5 × 10 ⁴ ) are placed inside the transwell and medium without serum (DMEM / F12, 100 units / ml penicillin-streptomycin) 100 Mix well in μl. Subsequent processes were performed in the same way as the invasiveness method.

As a result, the infiltration and mobility of HN and HCN were reduced compared to H, and among them, HN was further reduced than HCN (FIG. 7). This means that the 166-189 amino acids of H-Ras and the CAAX (H-Ras: CVLS, N-Ras: CVVM) sites are essential for invasiveness and mobility, and among them, the 166-189 amino acids of H-Ras protein are more important. It means to work. In addition, NH has increased invasiveness and mobility compared to N (FIG. 8), indicating that the 166-189th amino acid of the H-Ras protein is essential for invasiveness and mobility.

Figure 1 is a comparison of the amino acid sequence of H-Ras and N-Ras protein, "*" is a common amino acid of H-Ras / N-Ras, "Hypervariable" is a highly variable region of H-Ras / N-Ras , " ^◆ " indicates a palmitoylation site (Cys ¹⁸¹ , Cys ¹⁸⁴ ).

Figure 2 is a schematic of the structure of Ras proteins, 32-40 represents the effector binding domain, "*" represents the panesylation and palmitoylation sites, 100% identical to amino terminal 1 to 86 and , 87 to 165th have 85% homology, and 166 to 189th are highly variable regions.

Figure 3 schematically shows the structure of the fusion protein and mutant protein prepared in Example 1 of the present invention.

Figure 4 schematically shows the structure of the H-Ras / N-Ras fusion protein and the process of confirming it by DNA sequencing.

5 is a schematic diagram showing the structure of an H-Ras / N-Ras fusion protein or an N-Ras mutant protein (A), cytological morphology (B) and immunoblot analysis of cells transformed with the vector expressing the protein ( C) The picture shows the result.

6 is a schematic diagram showing the structure of N-Ras / H-Ras fusion protein or N-Ras mutant protein (A), cytological morphology (B) and immunoblot analysis of cells transformed with the vector expressing the protein ( C) The picture shows the result.

FIG. 7 is a graph showing the results of invasiveness (A) and �dynamics (B) studies of cells transformed with vectors expressing H-Ras / N-Ras fusion proteins or H-Ras mutant proteins. Invasiveness and mobility were significantly reduced in expressing transformed cell lines, and "**" indicates statistically different compared to the control group ( p <0.01, two-tailed Student's t test).

8 is a graph showing the results of invasiveness (A) and mobility (B) studies of cells transformed with a vector expressing an N-Ras / H-Ras fusion protein or an N-Ras mutant protein. Invasiveness and mobility were significantly reduced in the transformed cell line, and "**" was statistically different compared to the control group ( p <0.01, two-tailed Student's t test).

<110> Duksung Women's University Industry-Academic Cooperation Foundation <120> Ras Fusion Protein <130> 2007-11-29 <160> 29 <170> KopatentIn 1.71 <210> 1 <211> 189 <212> PRT <213> Homo sapiens <220> <221> DOMAIN (222) (166) .. (189) <223> hypervariable domain <220> <221> SITE (222) (32) .. (40) <223> effector binding site <220> <221> SITE <222> (181) <223> palmitoylation site <400> 1 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys   1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr              20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly          35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr      50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys  65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr                  85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val             100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys         115 120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr     130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp                 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met             180 185 <210> 2 <211> 189 <212> PRT <213> Homo sapiens <220> <221> DOMAIN 166 (166) .. (189) <223> hypervariable domain <220> <221> SITE (222) (32) .. (40) <223> effector binding site <220> <221> SITE <222> (181) <223> palmitoylation site <220> <221> SITE <222> (184) <223> palmitoylation site <400> 2 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys   1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr              20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly          35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr      50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys  65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr                  85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val             100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg         115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr     130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu                 165 170 175 Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser             180 185 <210> 3 <211> 189 <212> PRT <213> Homo sapiens <220> <221> SITE <222> (12) <223> mutation site <400> 3 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys   1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr              20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly          35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr      50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys  65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr                  85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val             100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys         115 120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr     130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp                 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met             180 185 <210> 4 <211> 189 <212> PRT <213> Homo sapiens <220> <221> SITE <222> (12) <223> mutation site <400> 4 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys   1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr              20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly          35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr      50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys  65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr                  85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val             100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg         115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr     130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu                 165 170 175 Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser             180 185 <210> 5 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fusion protein consisting of N-terminal amino acids of N-Ras          protein and C-terminal amino acids of H-Ras protein <220> <221> DOMAIN (222) (1) .. (165) <223> N-Ras part <220> <221> DOMAIN (222) (166) .. (189) <223> H-Ras part <220> <221> SITE <222> (12) <223> mutation site <400> 5 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys   1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr              20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly          35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr      50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys  65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr                  85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val             100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys         115 120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr     130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu                 165 170 175 Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser             180 185 <210> 6 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> fusion protein consisting of N-terminal amino acids of H-Ras          protein and C-terminal amino acids of N-Ras protein <220> <221> DOMAIN (222) (1) .. (165) <223> H-Ras part <220> <221> DOMAIN (222) (166) .. (189) <223> N-Ras part <220> <221> SITE <222> (12) <223> mutation site <400> 6 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys   1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr              20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly          35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr      50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys  65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr                  85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val             100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg         115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr     130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp                 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met             180 185 <210> 7 <211> 567 <212> DNA <213> Artificial Sequence <220> <223> a gene coding the fusion protein represented by SEQ. ID. No. 5 <400> 7 atgacggaat ataagctggt ggtggtgggc gccgatggtg ttgggaaaag cgcactgaca 60 atccggctaa tccagaacca ctctgtagat gaatatgatc ccaccataga ggattcttac 120 agaaaacaag tggttataga tggtgaaacc tgtttgttgg acatactgga tacagctgga 180 caagaagagt acagtgccat gagagaccaa tacatgagga caggcgaagg cttcctctgt 240 gtatttgcca tcaataatag caagtcattt gcggatatta acctctacag ggagcagatt 300 aagcgagtaa aagactcgga tgatgtacct atggtgctag tgggaaacaa gtgtgatttg 360 ccaacaagga cagttgatac aaaacaagcc cacgaactgg ccaagagtta cgggattcca 420 ttcattgaaa cctcagccaa gaccagacag ggtgttgaag atgcctccta cacgttggtg 480 cgtgagatcc ggcagcacaa gctgcggaag ctgaaccctc ctgatgagag tggccccggc 540 tgcatgagct gcaagtgtgt gctctcc 567 <210> 8 <211> 567 <212> DNA <213> Homo sapiens <400> 8 atgacggaat ataagctggt ggtggtgggc gccggcggtg tgggcaagag tgcgctgacc 60 atccagctga tccagaacca ttttgtggac gaatacgacc ccactataga ggattcctac 120 cggaagcagg tggtcattga tggggagacg tgcctgttgg acatcctgga taccgccggc 180 caggaggagt acagcgccat gcgggaccag tacatgcgca ccggggaggg cttcctgtgt 240 gtgtttgcca tcaacaacac caagtctttt gaggacatcc accagtacag ggagcagatc 300 aaacgggtga aggactcgga tgacgtgccc atggtgctgg tggggaacaa gtgtgacctg 360 gctgcacgca ctgtggaatc tcggcaggct caggacctcg cccgaagcta cggcatcccc 420 tacatcgaga cctcggccaa gacccggcag ggagtggagg atgccttcta cacgttggtg 480 cgtgagatcc ggcagcacaa gctgcggaag ctgaaccctc ctgatgagag tggccccggc 540 tgcatgagct gcaagtgtgt gctctcc 567 <210> 9 <211> 567 <212> DNA <213> Homo sapiens <400> 9 atgacggaat ataagctggt ggtggtgggc gccggcggtg ttgggaaaag cgcactgaca 60 atccagctaa tccagaacca ctttgtagat gaatatgatc ccaccataga ggattcttac 120 agaaaacaag tggttataga tggtgaaacc tgtttgttgg acatactgga tacagctgga 180 caagaagagt acagtgccat gagagaccaa tacatgagga caggcgaagg cttcctctgt 240 gtatttgcca tcaataatag caagtcattt gcggatatta acctctacag ggagcagatt 300 aagcgagtaa aagactcgga tgatgtacct atggtgctag tgggaaacaa gtgtgatttg 360 ccaacaagga cagttgatac aaaacaagcc cacgaactgg ccaagagtta cgggattcca 420 ttcattgaaa cctcagccaa gaccagacag ggtgttgaag atgcttttta cacactggta 480 agagaaatac gccagtaccg aatgaaaaaa ctcaacagca gtgatgatgg gactcagggt 540 tgtatgggat tgccatgtgt ggtgatg 567 <210> 10 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for HG12D protein (H) <400> 10 ctcggatcca tgacggaata taagctggtg ctgctgggcg ccgatggt 48 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for HG12D protein (H) <400> 11 agactcgagc gtcaggagag 20 <210> 12 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for NG12D protein (N) <400> 12 ctcggatcca tgacggaata taagctggtg ctgctgggcg ccgatggt 48 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for NG12D protein (N) <400> 13 agactcgagc gtcacatcac cac 23 <210> 14 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for N-terminal amino acids of HN fusion protein          (HN-1) <400> 14 ctcggatcca tgacggaata taagctggtg ctgctgggcg ccgatggt 48 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for N-terminal amino acids of HN fusion protein          (HN-1) <400> 15 ggcgatatcc tccactcc 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for C-terminal amino acids of HN fusion protein          (HN-2) <400> 16 gaagatatcg ctttttac 18 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for C-terminal amino acids of HN fusion protein          (HN-2) <400> 17 agactcgagc gtcacatcac cac 23 <210> 18 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for N-terminal amino acids of NH fusion protein          (NH-1) <400> 18 ctcggatcca tgacggaata taagctggtg ctgctgggcg ccgatggt 48 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for N-terminal amino acids of NH fusion protein          (NH-1) <400> 19 agcgatatct tcaacacc 18 <210> 20 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> forward primer for C-terminal amino acids of NH fusion protein          (NH-2) <400> 20 gaggatgcct cctac 15 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for C-terminal amino acids of NH fusion protein          (NH-2) <400> 21 agactcgagc gtcaggagag 20 <210> 22 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for HCN fusion protein <400> 22 ctcggatcca tgacggaata taagctggtg ctgctgggcg ccgatggt 48 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for HCN fusion protein <400> 23 agactcgagc gtcacatcac cac 23 <210> 24 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for NCH fusion protein <400> 24 ctcggatcca tgacggaata taagctggtg ctgctgggcg ccgatggt 48 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for NCH fusion protein <400> 25 agactcgagc gtcaggagag 20 <210> 26 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for H184N protein <400> 26 ctcggatcca tgacggaata taagctggtg ctgctgggcg ccgatggt 48 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for H184N protein <400> 27 agactcgagc gtcaggagag cacacacttc aag 33 <210> 28 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for N184H protein <400> 28 ctcggatcca tgacggaata taagctggtg ctgctgggcg ccgatggt 48 <210> 29 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for N184H protein <400> 29 agactcgagc gttacatcac cacacatggg cat 33  

Claims (12)

delete Ras fusion protein in the form of a combination of 1 to 120 amino acids of N-Ras protein and 121 to 189 amino acids of H-Ras protein. A Ras fusion protein in a form in which the 1-141th amino acid of the N-Ras protein and the 142-189th amino acid of the H-Ras protein are combined. delete The method according to claim 2 or 3, Ras fusion protein of the form wherein the 12th amino acid of the fusion protein is substituted with Asp in Gly. 1-n amino acid of the N-Ras protein and n + 1-189 amino acid of the H-Ras protein is combined form, wherein n is a natural number of 165 or less Ras fusion protein, the 12th amino acid of the fusion protein A Ras fusion protein having the amino acid sequence set forth in SEQ ID NO: 5 which is a form substituted with Asp in this Gly. A gene encoding a Ras fusion protein according to any one of claims 2, 3 and 5. A gene encoding a Ras fusion protein set forth in SEQ ID NO: 5. The method according to claim 8, A gene having the nucleotide sequence set forth in SEQ ID NO: 7. Recombinant expression vector comprising the gene of claim 7. A transformant transformed with the recombinant expression vector of claim 10. The method according to claim 11, A transformant in which the recombinant expression vector is introduced into MCF10A cells, which are breast epithelial cells.

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